January 15, 2014 (Vol. 34, No. 2)

From our January 15 issue: Processing in plastics is maturing, with users focusing on cell/product retention, rapid prototyping, and streamlined operations.

The confluence of several developments—expression system improvements, greater familiarity among regulators and drug makers, and component availability—has created a near-perfect environment for adopting single-use processing.

While the complications that remain have not yet been reduced to “engineering problems,” they are becoming more manageable. The industry has made great progress with processing in plastics, as indicated by several of the presentations at “BioProduction,” an Informa conference recently held in Dublin, Ireland.

Neil Ross, marketing manager of the bioprocess division at GE Healthcare Life Sciences, discussed cell- and product-retaining techniques in single-use “perfusion-like” intensified cultures. Where genuine perfusion cultures remove product continuously but retain cells, perfusion-like cultures have much greater leeway. Often, they may retain both product and cells, but allow low molecular weight waste products to pass through the membrane. “In certain respects, perfusion-like cultures resemble fed-batch more than standard perfusion cultures,” Ross said.

The main benefit of perfusion-like processes for the production of therapeutic proteins is that product does not become as dilute as in conventional perfusion cultures. Besides posing the challenge of continuously diluted product, more traditional perfusion has issues related to regulatory requirements and batch-to-batch variation. The two process types are not too dissimilar, however. Merely tweaking traditional perfusion allows one to approach perfusion-like processes.

Retention technology in perfusion processes may be filter- or gravity-based. GE Healthcare has several filter-based retention technologies. One example is cross-flow filtration with hollow fiber filters. Varying the hollow fiber membrane pore size from 0.65 micron (cell retention only) down to a 30 kD cutoff (retaining all significant species) is what has led to the development of perfusion-like cultures and their fed-batch like features.

Perfusion-like processes are capable of intensifying seed train operation. “By achieving much higher than normal cell density, operators can use much smaller seed train bioreactors, thus minimizing the number of steps to the bioproduction vessel and decreasing the investment for the overall manufacturing operation,” Ross explains

The technique may also cut time for media development because nutrients are constantly added and waste products continuously removed. Processors need not resort to extraordinary feed and media strategies to maintain the culture over many days. “Perfusion media is more forgiving than fed-batch,” Ross asserted.

The main challenges to perfusion cell culture in a single-use system have been the availability of high-performing, robust sensors and control systems suitable for cultures that run for up to 50 days, and maintaining sterility. According to Ross, new developments in single-use optical pH and dissolved oxygen sensors have eliminated one hurdle.

On the control side, GE Healthcare has created a version of its Unicorn™ software for upstream processing, while sterile connectors have greatly reduced the potential for contamination. In addition, GE Healthcare has different technologies for cell retention. One choice of perfusion membrane is a hollow fiber cross-flow filter. Unlike filters in cassette formats, hollow fibers are inherently low-shear because there is no restriction in the fluid path.

“During ultrafiltration, filters generate 10,000/sec of shear,” Ross explained. “But hollow fiber filters involve only about 500/sec. There’s less sweeping motion across the filter.” Filtration is run much more slowly than it would be in downstream purification—over the course of many days versus a few hours. Additional capacity is available by simply increasing the surface area.

After a purification step has been completed using ÄKTA ready, the single-use flow paths (i.e., ÄKTA ready flow kits and gradient flow sections) are disposed. [GE Healthcare]

Rapid Prototyping

The prototyping of single-use systems and components represents a significant holdup in process design. “Customers are under increasing pressure to complete development work and bring products to market,” said Chris Shields, marketing manager for single-use products at Saint Gobain Life Science. “It sometimes takes months to reach the prototype stage.” Often, he says, customers that cannot purchase a ready-made system or its components will piece together makeshift systems.

At “BioProduction,” Shields described Saint Gobain’s rapid prototyping of single-use components that practically assures delivery of any part or system within weeks. This capability arises from the company’s expertise in high-performance plastics for a wide range of industries.

Customers use an online design tool based on the leading 3D rendering software, provided by SolidWorks, and off-the-shelf web hosting software. “Customers can work directly with our design engineers within the software to arrive at a single-use process design in less than an hour,” Shields added.

Using one of its 3D printers, Saint Gobain can also produce prototype parts for evaluation. If the part is suitable, it will cut a metal dye and produce the part. “We take a prototyping process that can last up to six months, and potentially shorten it to two weeks,” Shields said. Saint Gobain can work with processes that scale as high as 2,000 L.

Within the SolidWorks system, Saint Gobain has built an in-house component validation library. The system reveals all certifications and validation information for any component selected, such as whether it is animal component-free or gamma-irradiated. The library includes Saint Gobain parts as well as those from competitors because, as Shields pointed out, “nobody in this industry has every component.” According to Shields, the program has been “a phenomenal success” that has enabled quote win rates four times higher than previously.

Automation: Why Not?

As single-use biomanufacturing moves forward, it has taken on qualities of multi-use processing that are useful, while discarding those that are not. As Pauline Nicholson, Ph.D., global product manager for single-use systems at Pall, has demonstrated, automation clearly falls into the “useful” but under-utilized category for disposable processing.

This trend is a function of the evolving complexity of disposable systems. “Customers are moving beyond simple bag and filter technologies into single-use downstream processing that includes tangential flow filtration (TFF), virus filtration, and other operations—which, in a multiple-use facility, would employ automation,” Dr. Nicholson added. “People used to believe that single-use processing was exclusively manual, where in fact it’s possible to apply to it many of the automation features of stainless steel processes.”

Automation involves the use of software-based control systems, sensors, and other hardware and software that allows walk-away operation. Pre-programmed recipes, for example, bring the same value to single-use processes as they do for multiple-use processes: consistency, less opportunity for operator error, and ultimately improved product quality. Typically, automated unit operations occur with the help of fixed-equipment process skids, controllers, and computers serving completely disposable fluid paths.

“There’s a diversity of operations in any process,” Dr. Nicholson explained. “The simplest ones do not require automation, but others, while straightforward, are repetitive and predictable.” Examples include buffer prep and pH adjustment. “Then there are other steps where it’s critical that the steps optimized during process development are run the same every time.”

These steps, Dr. Nicholson says, are ideal candidates for automation. Instead of an operator following a written standard operating procedure, the protocols upload into the control system, which automatically runs the steps. Many if not most processes, Dr. Nicholson said, are translatable into a series of automated steps.

Crucial to automating single-use processes are skids with the right mix of non-disposable components and single-use product flow paths. These tend to be smaller and more versatile than skids designed for fixed-tank operation. Pumps, pressure transmitters, flow monitors, and instrumentation remain, but once the need for cleaning has been removed, the skids can be more compact and versatile; one can handle several unit operations.

“For, say, TFF, skids can be a lot smaller than for an equivalent stainless-steel system. You don’t need to include a lot of piping for cleaning,” Dr. Nicholson asserted.

Pall says it designed the Allegro MVP system for flexible automation in both upstream and downstream single-use processes.

“Leaning” the Process

The multiple steps normally involved in media reconstitution from dry ingredients raise several issues within a single-use process. Generally, long culture times produce highly cell-dense cultures that require feeding, often through a combination of multiple components.

Media and feed operations must therefore evolve, says Cynthia Hoy, Ph.D., a process science fellow at Life Technologies, to avoid becoming a process bottleneck. “We’re reaching the solubility limits for many of these components,” Dr. Hoy noted. “Often they cannot exist in the same solution simultaneously at the necessary concentrations.”

Bioprocessors can apply quick fixes such as altering the pH or adding feed components rapidly, but these remedies complicate an already intense production workflow. Life Technologies’ solution is to simplify the process by offering dry media and feed as a single “part” consisting of its many diverse components.

The issue here is not any special circumstance brought about by single-use processes: Media and feeds are used interchangeably between stainless-steel and single-use processes. What’s at stake is harmonization of all steps with single-use processes that promise simplicity and versatility.

“You’re defeating the purpose of streamlining a plant through single-use if complicated feeds require long mixing times at extreme pH that may be incompatible for both stainless and resins,” Dr. Hoy observed. “Feed delivery could become the unplanned bottleneck.”

At “BioProduction,” Pall introduced a revamped prototype product that increases the effective feed concentration delivered to cells. Part of this strategy involves minimizing dilution within the bioreactor, and thus taking greater advantage of the available culture volume. “This idea is important in stainless-steel systems, but it is especially so in single-use systems which tend to be smaller than stainless,” Dr. Hoy concluded.

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